The separation and purification of complex molecules of biological interest has been the subject of extensive research. Since many of these molecules are ampholytes, they are theoretically amenable to separation by electrophoretic techniques. A mixture of ampholytes in solution, when subjected to a direct current electric potential will arrange within this electric potential according to isoelectric point, thereby resulting in separation.
In recent years, an improved technique for separation of ampholytes, known as isoelectric focusing, has been developed. According to this technique, an artificial pH gradient is established by use of a mixture of synthetic compounds (referred to as carrier ampholytes). Upon electrophoresis of an ampholyte mixture in such a pH gradient, which acts as buffer, the ampholytes arrange themselves within this gradient. In essence, each ampholyte molecule migrates towards the pH value in the gradient where it is isoelectric, that is, where its net charge is 0. The focusing thus takes place at the point where the pH is equal to the pI of the ampholyte.
Detailed treatment of the theory and techniques of isoelectric focusing may be found in Haglund, Methods of Biochemical Analysis, Vol. 19, page 1; and Annals of the New York Academy of Sciences, Vol. 209, "Isoelectric Focusing and Isotachophoresis".
Recently, a series of devices for use in isoelectric focusing have been described in Valmet in U.S. Pat. No. 3,616,456 and Science Tools, Vol. 16, page 1 (1969). These devices have a series of chambers (usually "U-tubes" or "V-tubes") disposed one after the other. The advantage of such a system is that if contamination occurs in one chamber, for example by precipitation of a solid, this does not result in contamination of the entire system. These devices were described as operating at relatively low voltages, from about 800 to 2400 volts, and with a power input of about 15 to 20 watts for a 45 ml. unit. Under these conditions, there is a limitation upon the resolution, the capacity, and, particularly, the speed of an electrophoretic separation since speed is directly proportional to the applied voltage and resolution is proportional to the square root of the voltage. The time factor becomes critical when one is dealing with sensitive ampholytes.
Thus, it would be desirable to develop techniques, and devices therefor, to allow for preparative separation of ampholytes with high resolution, at high speed, and with great reproducibility.